The present invention relates to a method for separating polypeptides (e.g. proteins), and more particularly to a method for separating a first polypeptide fraction from a second polypeptide fraction of a mixed solution containing the fractions by selective precipitation of one of the fractions.
Single-phase methods disclosed heretofore for purifying proteins using polymeric precipitants fall into two general methods which are both based on differential solubility of proteins. In one method, a neutral polymer is added to the protein solution to cause a decrease in the solubility of the target protein which therefore precipitates out of solution. By "target protein" is meant the protein or protein fraction that is precipitated from solution as a result of its interaction with a polymeric precipitant. Neutral polymeric precipitation methods have severe disadvantages. Precipitation using neutral polymers is not usually very selective. Use of neutral polymeric precipitants often requires that the solution pH be near the isoelectric point of the target protein to obtain significant precipitation. In addition, this method is not applicable to dilute protein solutions since relatively high concentrations of both protein and neutral polymer are normally required for precipitation to occur.
In the other method, a charged polymer is used to form a complex with the target protein. The complex, which is usually much less soluble than the uncomplexed target protein, precipitates out of solution. While charged polymeric precipitants are more specific with respect to the target protein and require only stoichiometric amounts of polymer, precipitation often occurs only in a very narrow range of operating conditions, i.e. pH, protein concentration and polymer concentration. In addition, precipitation is reduced in the presence of chaotropic agents such as urea.
Methods have also been disclosed for protein purification using aqueous two-phase systems. These methods are based on the incompatibility of polymers in aqueous solution and the partition coefficient of the target protein versus other contaminating proteins. The reason for the incompatibility is believed to be the inability of the polymer coils to penetrate each other. As a result, if one mixes polymer A with an incompatible polymer B at sufficiently high polymer concentration an aqueous two-phase system can be formed. Likewise, aqueous multiple-phase systems can be obtained by mixing several incompatible polymers at sufficiently high concentration. The polymers for these aqueous multiple-phase systems are chosen such that the solubility of the target protein is much higher in one polymer phase than the others. The partition coefficient can be somewhat enhanced by the addition of small amounts of salts or polyelectrolytes. Unfortunately, multi-phase methods require separation of the protein-rich phase and subsequent recovery of the protein therefrom.
It has now been found that the effectiveness of polypeptide separation using charged polymeric precipitant can be enhanced by using, in addition to the charged polymer, at least one soluble neutral polymer. The effectiveness of the charged polymer may be enhanced by improving or otherwise modifying the precipitation of the target fraction and/or the selectivity of the charged polymeric precipitant for the target polypeptide fraction.
It is, therefore, the overall object of the present invention to provide an improved method for separating polypeptides.
Accordingly, it is an object of the present invention to provide a separation method capable of high yields.
It is another object of the present invention to provide a separation method capable of high selectivity.
It is yet another object of the present invention to provide a method capable of operating at a low target polypeptide concentration.
It is still another object of the present invention to provide a separation method capable of operating at a relatively low charged polymer concentration.
It is yet another objective of the present invention to provide a separation method capable of operating under a wide range of conditions in terms of pH and materials present such as chaotropic agents, etc.
These and other objects and advantages of the present invention will be evident to those skilled in the art from the following description and examples.